For treating or examining cells in molecular biology or genetic engineering, devices are used which consist, on the one hand, of a vessel (for example a Petri dish) for receiving the cells and a reaction liquid, and, on the other hand, of a cover (xe2x80x9cLab-Tek IIxe2x80x9d brochure from Nalge Nunc International, Naperville; xe2x80x9cEasiSealxe2x80x9d brochure from HYBAID Limited, Teddigton; xe2x80x9cGene Framexe2x80x9d brochure from Advanced Biotechnologies Ltd., Epsom; EP-A 611 598). A distinction must be made here between covers which are simply placed loosely on the upper edge of the vessel and airtight lids which are connected adhesively and airtight to the edge of the device or to a slide forming the base of the device. When the latter covers are applied, care must be taken to ensure that air bubbles are avoided between the lid and the substrate. This is generally done by means of some of the reaction liquid being displaced towards the side as the flat lid is being applied. However, this is only possible if the vessel has no walls. As these are desired in certain process stages, walls are provided which can be tightly connected to and detached from the base (slide) of the device. In the known devices mentioned above, the possibility of removal of the walls is also provided because a substrate layer which is so thin that the temperature changes needed during the process can be effected quickly and with precision control can be easily produced with a flat lid.
Vessels with vertical walls are also known in which a thin substrate layer is produced by means of a cover being lowered partially into the vessel (U.S. Pat. No. 4,294,924; U.S. Pat. No. 4,321,330; DE-A-196 24 917). The thin substrate layer is enclosed between a base of the cover and the base plate of the vessel. The cover is inserted into the vessel, with air and excess liquid being displaced. This makes tight closure difficult.
The invention is based on the object of providing a method and a device which permit the optionally thin-layered substrate to be sealed off even in cases where walls are present. The solution lies in the features of claims 1 and 2.
When the cover is being introduced between the walls of the vessel, the air situated above the substrate in the vessel is displaced through the outflow opening. A possible excess amount of reaction liquid is also displaced through the outflow opening. The cover can be lowered inside the vessel until the desired thickness of the substrate layer is achieved between the base plate of the vessel and the opposite cover base, which desired thickness can range from several hundredths of a millimetre to several millimetres.
In order to avoid undesired clearance, the circumferential surface of the cover can fit closely, with an essentially identical shape, to the internal surface of the walls. This fit can also constitute the sealing of the cover with respect to the walls, for example by means of a ground-glass joint or a plastic seal under elastic pressure. However, this does not need to be the case, since, in addition to a close, but not tight fit of the circumferential surface of the cover to the internal surface of the walls, a special seal can be provided which can cooperate, for example, with the edge of the vessel walls or a shoulder thereof. In these cases it is often expedient to provide a holder for maintaining the sealing position of the cover with respect to the vessel, for example a screw-on, snap or spring closure; however, the tight fit of the cover on the vessel walls can also be self-supporting, for example as a result of the frictional forces which exist between two ground-glass surfaces.
It is expedient for the lower surface of the cover base to extend approximately parallel to the base of the vessel in order to be able to achieve an essentially constant layer thickness. The cover and the vessel can be provided with -cooperating contact surfaces which define the size and constancy of the layer thickness.
The outflow opening can be provided with a closure device. However, the possibility also exists of providing sealing by means of a subsequently applied oil layer. This also applies to the circumferential sealing. In many cases a tight seal is only required in respect of the external atmosphere in order, for example, to avoid evaporation or the admission of oxygen. In these cases it may suffice if the seal is formed not on the vessel and cover, but is instead formed by an apparatus which receives the device, for example a type of autoclave in which there is an internal atmosphere which is chosen in accordance with the intended purposes. To avoid evaporation, for example, it can have sufficient moisture. To avoid admission of harmful gases, it can consist of nitrogen or noble gas.
To ensure that it is not only the air in the area of the outflow opening that is removed, but also that air quantity which may be contained between the circumferential surface of the cover and the vessel walls, it is possible for the outflow opening to be provided with an ascending section whose mouth is at least approximately level with the upper end of the gap situated between the circumferential surface of the cover and the vessel walls. On slow insertion of the cover into the vessel, the static pressure of the liquid column present in the ascending section of the outflow opening then ensures that the air in the circumferential gap is also displaced.
When the cells have deposited sufficiently firmly on the base of the vessel, the excess liquid can be readily drawn off or displaced through the outflow opening without any risk of losing cells. So that it is also possible to work with cells which are in suspension, the outflow opening can be provided with a screen which holds back the cells during displacement of the liquid.
A plurality of openings can be provided which are designed for attachment of an admission and discharge line for a medium. One or more of these openings can serve as outflow opening. The device can then also be used as a so-called reactor (Meenen et al.; xe2x80x9cSemi Continuous Reactor System . . . xe2x80x9d, Poster 1994 Biomaterials 21:905-908).
The cells involved can be adherent cells which stick to the base of the cell culture dish, or suspension cells which swim in the culture medium. Alternatively, tissue sections can also be cultured in the dish. In general, both eukaryotic and prokaryotic cells can be cultured in the vessel. Treatments and examinations which can be carried out using the device according to the invention can, for example, be all types of in situ hybridization and in situ PCR.
The outflow opening is expediently provided inside the cover, at a distance from the edge of the latter. However, this does not exclude the possibility of its being formed by means of a spacing provided, at least in places, between the edge of the cover and the vessel walls. A particularly advantageous design in this connection is one in which the edge of the cover comprises a collar which rises upwards from the bottom of the cover plate, which forms in its entirety or in places the said spacing for forming an outflow opening, and whose upper edge cooperates with the vessel walls to form a seal. As the cover is being lowered, quantities of gas and liquid enclosed between cover and vessel can then escape, and it is only at the end of this procedure, when the cover reaches its end position, that the seal is obtained.
If the intention is to subject a plurality of cultures to the same thermal conditions, it is expedient to connect a plurality of vessels to one another. In this case, a cover of the type indicated above can be provided for each one of the vessels. However, it is also possible to connect a plurality of covers to one another for joint actuation. It is of course possible to make the walls of a plurality of vessels integral with one another, in which case either the bases are also connected in one piece with the walls or they can be separated from these, for example in the form of a slide.
Of particular importance, however, in the context of the invention is the possibility of being able to create a plurality of chambers by means of forming walls inside one and the same vessel, which chambers, during the course of treatment of a culture, are at times separated from one another, or are not separated, depending on the requirements. For this purpose, the cover is provided with an arrangement for dividing off such chambers from one another. A particularly advantageous design is one in which a narrower vessel is so designed that it can be accommodated in a wider vessel. The cover of the wider vessel is in this case generally designed in such a way that it serves solely to close the area of the wider vessel situated outside the narrower vessel. For the closure of the narrower vessel, a special closure is then optionally provided, for which the general comments made above in respect of the closure of the vessels also apply. This means that in addition to a simple air-permeable or airtight closure, a cover can also be provided which is equipped with an opening for displacement of the atmosphere, and possibly excess liquid, arising in the narrower vessel, and whose air opening can be closed. However, there is also the possibility of structurally connecting the covers of the wider vessel and of the narrower vessel to one another for joint actuation.
Where, in the present connection, mention is made of xe2x80x9caxe2x80x9d narrower vessel, this is intended to mean that it is at least one; a plurality of narrower vessels can also be provided, however, in a wider vessel. The arrangement of a narrower vessel in a wider vessel affords the possibility of subjecting the cultures within the narrower vessel and within the wider vessel to exactly the same thermal conditions. They can have separate bases. Of particular importance, however, is a design in which the narrower vessel shares the base with the wider vessel. This affords the possibility of subjecting one and the same culture inside the wider vessel to different reaction conditions (for example another reaction liquid) during all or some of the reaction steps. Accordingly, the wall of the narrower vessel is used only in those reaction steps, inside the otherwise uniform culture, in which the reaction conditions are intended to be different, e.g. for positive control or negative control in PCR.
There are cases in which the narrower vessel can remain in the wider vessel during the entire reaction or sequence of reactions. In these cases, it can be permanently connected to the base and to the wider vessel from the outset at the factory stage. In other cases, the compartmentalization is desired at the start of the reaction or chain of reactions; the narrower vessel can then be connected, likewise at the factory stage, to the base of the wider vessel, but it can be detached therefrom so that the compartmentalization can be annulled at the desired time. The factory-stage arrangement has the advantage that the narrower vessel can be accurately positioned in relation to the walls of the wider one, and this therefore guarantees that the cover fits. In other cases, one will want to have the freedom to be able to carry out the compartmentalization at any desired time. In these cases, the wall of the narrower vessel can be fitted by the user.
If the cover is intended to close off the wider vessel completely, it must not only have its circumferential surface adapted to the wall of the wider vessel, but must also be able to be connected sufficiently tightly to the narrower vessel. This can be achieved by its having a cutout which is adapted to the shape of the narrower vessel and which can join to the latter with the desired tightness. This cutout can be formed by an outflow opening. In another embodiment, the cover is connected in one piece to the wall of the narrower vessel, so that the compartmentalization made possible by the narrower vessel comes about with the insertion of the cover. In both cases, the cover serves as a holder or guide during insertion and, if appropriate, also during use of the narrower vessel.
In some cases an absolutely tight seal between the wall of the narrower vessel and the base of the wider vessel is not necessary; the narrower vessel can then simply be formed by a small tube which is held by the cover and reaches down to the base of the wider vessel. If a tight connection of the narrower vessel wall against the base is required, the lower edge of the wall of the narrower vessel can be provided with a seal, the wall being pressed against the base by the cover or an additional clamp in order to generate the sealing pressure. The wall of the narrower vessel can also be bonded to the base. In the simplest case, the vessel wall of the narrower vessel consists of a sealing ring which is fitted between the base plate and the cover base, fits tightly against these and divides the chamber enclosed by it from the space surrounding it. To ensure that the enclosed chamber can be subjected to a different treatment, it must be accessible by way of a cover opening which is located in its area and which can be the outflow opening.
According to the invention, the cover can also be provided with further arrangements which are desired for the treatment of the cultures. For electroporation, the cover and the base can support electrodes, for example planar electrodes, and the cells to be treated are arranged on a porous membrane between these.
If displaced liquid is to be collected, or if a reservoir of liquid is to be formed which is intended to replace the liquid present in the treatment chamber, a trough can be formed on the upper side and communicate with a corresponding opening in the cover. For liquid which is to pass into the treatment chamber by diffusion or flow, the trough base should not be lower than the mouth of the opening. A plurality of openings can be provided per vessel in order to permit liquid exchange by flow. For this purpose, a plurality of openings should be provided, of which at least one is connected to a trough which is separate from the trough or troughs of the other openings and which is not deeper than the mouth of the opening.
Particularly with respect to machine treatment of the devices, it may be expedient to combine a plurality of vessels and provided them with a common cover which has the requisite openings in the area of each individual vessel.
For machine treatment, a machine is provided which is equipped with means for introducing at least one liquid into at least one of the troughs or openings. It can also be designed for suctioning liquid from a trough or opening.
In this context, the term trough is intended to signify any vessel-like depression without stating its width or height, unless such a statement is expressly made. The subjects of claims 23 to 26 and of FIGS. 12 to 16 serve for protection independently of claims 1 to 22.